ABSTRACT: The endothelial cell (EC) cytoskeleton is a complex array of proteins intimately involved in the diverse cell shape changes critical to dynamic regulation of lung vascular barrier integrity, particularly in the pathobiology of and recovery from ARDS and VILI. Consistent with the thematic underpinnings of this PPG, Project #2 investigators have identified specific cytoskeletal effectors (cortactin, Ena/VASP-like protein or EVL, c-Abl) involved in the actin-based cytoskeletal remodeling that is spatially defined at the EC periphery and within lamellipodia protruding into the paracellular space. Complementing studies described in Project #1, Specific Aim #1 will focus on Arp2/3-mediated actin polymerization dynamics in vitro and the effects of c-Abl-mediated post-translational modifications (PTMs, tyrosine phosphorylation) and cytoskeletal effector coding polymorphisms (SNPs) on rates of actin polymerization. Specific Aim #2 will next define the critical participation of key cytoskeletal effector proteins (cortactin, EVL, c-Abl) in regulation of peripheral actin cytoskeletal remodeling, and EC barrier responses (lamellipodia formation, paracellular gap closure, barrier restoration). In addition, the functional contribution of specific cytoskeletal effector PTMs, including c-Abl- mediated cytoskeletal protein tyrosine phosphorylation, will be assessed. These studies will employ Core D expertise and utilize mutant fusion proteins, live cell imaging with kymography, super resolution, atomic and traction force microscopy, protein-protein binding assays, and intercellular gap closure assays to visualize the contributions of these peripheral cytoskeletal effector proteins to EC barrier responses. Similarly, Project #2 investigators have preliminarily defined key non-synonymous coding SNPs that significantly influence protein functionality in peripheral remodeling and EC barrier restoration. Specific Aim #3 will interrogate the influence of cytoskeletal effector coding SNPs that we speculate affect cytoskeletal effector protein function either via altered protein-protein interactions (cortactin-EVL interaction) or altered kinase activity (c-Abl) within the peripheral actin network. These studies, designed to define the functional consequences of specific coding SNPs within each cytoskeletal effector protein, will use the array of biophysical and imaging modalities described for SA #2. Finally, utilizing preclinical models of ARDS and VILI (Core C), Specific Aim #4 will characterize the in vivo influence of cytoskeletal effector protein PTMs and SNPs that we have determined to be impactful in vitro. Based on our compelling published and preliminary data that strongly indicate that each cytoskeletal effector protein to be studied in Project #2 is required to produce agonist-induced lung EC barrier enhancement, Project #2 cytoskeletal effectors are attractive molecular targets for novel therapies and warrant intense structure/function investigation.